Extracellular microRNAs in human circulation are associated with miRISC complexes that are accessible to anti-AGO2 antibody and can bind target mimic oligonucleotides

Abstract

MicroRNAs (miRNAs) function cell-intrinsically to regulate gene expression by base-pairing to complementary mRNA targets while in association with Argonaute, the effector protein of the miRNA-mediated silencing complex (miRISC). A relatively dilute population of miRNAs can be found extracellularly in body fluids such as human blood plasma and cerebrospinal fluid (CSF). The remarkable stability of circulating miRNAs in such harsh extracellular environments can be attributed to their association with protective macromolecular complexes, including extracellular vesicles (EVs), proteins such as Argonaut 2 (AGO2), or high-density lipoproteins. The precise origins and the potential biological significance of various forms of miRNA-containing extracellular complexes are poorly understood. It is also not known whether extracellular miRNAs in their native state may retain the capacity for miRISC-mediated target RNA binding. To explore the potential functionality of circulating extracellular miRNAs, we comprehensively investigated the association between circulating miRNAs and the miRISC Argonaute AGO2. Using AGO2 immunoprecipitation (IP) followed by small-RNA sequencing, we find that miRNAs in circulation are primarily associated with antibody-accessible miRISC/AGO2 complexes. Moreover, we show that circulating miRNAs can base-pair with a target mimic in a seed-based manner, and that the target-bound AGO2 can be recovered from blood plasma in an ∼1:1 ratio with the respective miRNA. Our findings suggest that miRNAs in circulation are largely contained in functional miRISC/AGO2 complexes under normal physiological conditions. However, we find that, in human CSF, the assortment of certain extracellular miRNAs into free miRISC/AGO2 complexes can be affected by pathological conditions such as amyotrophic lateral sclerosis.

Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin

“…Importantly, as shown by Crescitelli et al ., (Crescitelli et al., 2013 ) EVs derived from different cell lines also have different RNA profiles, thus, the EV RNA profile from conditioned cell medium shown in this study is specific for MM6‐derived EVs. Importantly, also “free” extracellular RNA, for example, RNA associated with Argonaut 2 complexes or ribonucleoprotein complexes, can be co‐isolated together with the EV fractions, which can also play a role in the difference in RNA profiles seen by the different methods (Galvanin et al., 2019 ; Geekiyanage et al., 2020 ).…”

Isolation of Cell-Free miRNA from Biological Fluids: Influencing Factors and Methods

“…References Membrane-coated microparticles Apoptotic bodies (0.1-5 µm) [15][16][17] Microvesicles (>0.5 µm) [17][18][19][20][21][22][23] Ectosomes (100-600 nm) [24] Oncosomes (1-10 µm) [17,25,26] Exosomes (30-150 nm) [17,18,22,24,[27][28][29][30][31][32][33]] Specific microvesicles (prostasomes (50 nm-0.5 µm), melanosomes (>0.5 µm), 'platelet dust' (~130-500 nm)) [33][34][35][36][37] Membrane-free microparticles High-density lipoproteins (HDL) [23,[38][39][40][41][42] Low-density lipoproteins (LDL) [39,40,42] Various RNA-binding proteins (for example, AGO1, AGO2, nucleophosmin 1 (NPM1), Tamm-Horsfall protein (THP), etc.) [23,[43][44][45][46][47][48]…”

“…[17,18,22,24,[27][28][29][30][31][32][33]] Specific microvesicles (prostasomes (50 nm-0.5 µm), melanosomes (>0.5 µm), 'platelet dust' (~130-500 nm)) [33][34][35][36][37] Membrane-free microparticles High-density lipoproteins (HDL) [23,[38][39][40][41][42] Low-density lipoproteins (LDL) [39,40,42] Various RNA-binding proteins (for example, AGO1, AGO2, nucleophosmin 1 (NPM1), Tamm-Horsfall protein (THP), etc.) [23,[43][44][45][46][47][48][49] Exomeres (~35 nm (<50 nm)) [50,51] miRNAs associated with the surface of blood cells [27,32]…”

“…It is therefore not surprising that cf-miRNA in supernatants of MCF-7 cells, blood [ 43 ], urine [ 75 ] and pericardial fluid [ 76 ] were found in strong complexes with AGO2 (Kd ~20–80 nM) [ 77 , 78 , 79 ]. qRT-PCR profiling of 375 miRNAs in size-exclusion chromatography fractions of human plasma more than 67% of the assayed miRNAs were associated with fractions containing AGO2 protein and a corresponding portion of plasma miRNAs could be recovered by AGO2 immunoprecipitation from plasma [ 47 ]. In addition to AGO2, miRNA in blood can be in a complex with AGO1 [ 45 ].…”

isomiRs–Hidden Soldiers in the miRNA Regulatory Army, and How to Find Them?

“…Several studies on body fluids such as blood (plasma and serum), urine, sputum, cerebrospinal fluid, and milk have shown an abundance not only of numerous miRNAs but also of their isomiR variants [ 123 ]. Although many of them can be found in circulation [ 124 ], enrichment of miRNAs and their isomiRs can be found in blood analytes such as human platelets (e.g., cytoplasm fragments derived from bone marrow megakaryocytes) and extracellular vesicles (EVs, e.g., secreted membrane-enclosed particles that are released naturally from the cell) [ 70 , 120 , 123 , 125 ]. In addition, EVs isolated from the urine of prostate cancer patients showed enrichment in miRNAs and isomiRs with potential for biomarkers in minimally invasive diagnosis of prostate cancer patients [ 126 ].…”